1. Field of the Invention
This invention relates to the use of 2,3,5,6-tetrahydroxy-1,4-quinone and related compounds as an activator for intrinsic blood coagulation and for use as a diagnostic reagent for the activated partial thromboplastin time test of blood coagulation.
2. Description of Related Art
The activated partial thromboplastin time (APTT) test measures the clotting time required for blood coagulation of the intrinsic blood coagulation pathway. The blood coagulation mechanism is a complex series of interrelated protein reactions which may be thought of as occurring in a "cascade" effect. The last stares of the cascade involve the formation of a visible blood clot (whole blood trapped in a mesh of fibrin strands). The intrinsic system's pathway utilizes constituents which are present in the blood plasma. As is known to those skilled in the art, the clotting of plasma in vertebrate blood centers around the fibrinogen molecule, a large protein (MW 340,000 in the human species) with a disulfide bridged doublet structure three different constituent chains: (.alpha..beta.B ).sub.2. Another substrate known as "cold insoluble globulin" (MW 400,000) may also be involved by helping to anchor the fibrin network to the surface of fibroblasts. The clotting of fibrinogen is controlled in a consecutive manner by two different enzymes, thrombin (factor II.sub.a, a serine protease) and fibrin stabilizing factor, a transamidase with a cysteine-SH active center (factor XIII.sub.a). In the intrinsic pathway, the chain of events leading to coagulation is triggered by the exposure of plasma to nonendothelial surfaces such as glass in vitro.
The protein components of the pathway include zymogens (Factors XII, XI, IX, X, II and XIII) and regulatory proteins concerned with activation (Factors VIII and V) or inhibition processes ("antithrombin III")). In general, the regulatory proteins of activation require the participation of phospholipids (e.g. cephalin, phosphatidylethanolamine, lecithin) and of calcium (Ca.sup.2+) ions.
The first phase in the intrinsic mechanism is the activation of Factor XII. The major function of the activated Factor XII, Factor XII.sub.a, is the activation of the plasma thromboplastin antecedent (PTA, Factor XI) which, with the presence of ionic calcium, activates another coagulation component (Factor IX). The combination of activated Factor IX, Factor VIII.sub.a and of platelet factor 3 (a phospholipid), forms an agent which activates the Stuart-Prower factor (SPF, Factor X). The activated SPF in the presence of the labile factor (Factor V) form a complex which catalyzes the conversation of prothrombin (Factor II) to thrombin in the presence of ionic calcium.
Thus, after the initial interaction of tissue, platelet and plasma factors, prothrombin is activated to thrombin, a hydrolytic enzyme of great specificity. Thrombin (Factor II.sub.a) brings about the conversion of fibrinogen to fibrin and it also regulates the rate of formation of fibrin stabilizing factor (Factor XIII.sub.a), a transaminating enzyme which cross-links fibrin.
The APTT test has been used to detect disorders of the intrinsic blood coagulation pathway and to monitor patients undergoing anti-coagulation therapy. In the APTT test, an activator compound, a phospholipid and calcium ions are added to the plasma being tested. The time for the plasma to clot is measured from the time the calcium ions are added until the start of coagulation. If there are deficiencies in any of the various factors making up the blood coagulation mechanism, these deficiencies are manifested in abnormal clotting, e.g., failure to clot, excessive prolonged clotting times, or prolonged clot retraction.
There are a number of activators for the APTT test, including silica, kaolin, Ellagic acid and sufatides. See Babson, U.S. Pat. No. 3,880,714 (1975), Speck, U.S. Pat. No. 3,486,981 (1969), and Witt, U.S. Pat. No. 4,672,030 (1987). However, silica and kaolin are heavy particles and tend to "settle" in the test sample during the coagulation assay in the automated instrument. That is, the silica and kaolin will fall out of the solution and the results of the assay may be erroneous.
Use of Ellagic acid-based reagents have given varying results in the APTT test. Ellagic acid has two internal ester bonds which may undergo hydrolysis in an alkaline pH medium. Without being limited by theory, it is thought that the variations in results of the APTT tests using Ellagic acid may be due to a structural change of the Ellagic acid in the reagent.
Sulfatides have been tested by applicants herein, according to the method described in Witt. At 100 ug/ml, the clotting time was 47 seconds, indicating a low level of activity. In addition, sulfatides are approximately 80 times more expensive than the activator of the present invention.
In Japanese patent No. 60-174952 (1985), it is disclosed that orthoquinone compounds coagulated plasma in approximately 5 to 8 minutes. However, the orthoquinone compounds could not be used in an aqueous medium because they are extremely unstable in an aqueous medium. Thus, they must be prepared and used in a water free, organic medium. (See, e.g. Fieser and Fieser, Organic Chemistry, 3rd Ed. at p. 716 (D. C. Heath and Company, Boston), and Chem. Ber 37, 4744-4746 at 4746 (1904).) Hence the orthoquinone compounds coagulate only a very small portion of the proteins available in plasma for coagulation. Moreover, the coagulation time disclosed in Japanese patent No. 60-174952 is several minutes longer than that of the present invention. The paraquinone of the present invention thus has a much high level of clotting activity.